T-lymphocytes, or T-cells, are cornerstones of the mammalian immune system, responsible for cell-mediated immunity against foreign antigens. T-cells, like other lymphocytes, develop from pluripotent hematopoietic stem cells produced primarily in hematopoietic tissues (i.e. in the liver in fetuses and in bone marrow in adults). Some of these precursor stem cells migrate through the blood to the thymus, and T-cell differentiation occurs there.
The majority of T-lymphocytes are immune system regulators, known as helper T cells and suppressor T cells, that act either to enhance or suppress the immune responses of other white blood cells. Other T-lymphocytes, called cytotoxic T-cells, act to kill virus-infected cells. These different types of T-cells are distinguished from one another by the presence of different antigenic markers on their surfaces. Specifically, helper T-cells express a cell-surface glycoprotein known as CD4, and cytotoxic T-cells express a different cell-surface glycoprotein, CD8. As T-cells mature, they express the CD2 cell-surface protein, and, ultimately, the CD3 cell-surface protein complex.
T-cell development has been intensively studied in vivo. The evidence indicates that pluripotent hematopoietic stem cells are present in populations of cells expressing CD34 surface molecules (CD34.sup.+ cells; see, for example, Terstappen et al. Blood, 79:666-677, 1992 and references cited therein). Such CD34.sup.+ cells represent about 1% of nucleated bone marrow cells. CD34.sup.+ cells have been used to successfully repopulate the thymus of an irradiated host with T-cells (see, for example, Exine et al. Nature 309:629-632, 1984; Goldschneider et al. J. Exp. Med. 163:1-17, 1986; Berenson et al. J. Clin. Invest. 81:951-955, 1988); and engraftment of human CD34+ progenitor cells into mice with genetically determined severe combined immunodeficiency (SCID mice) has been observed to result in development of mature T-lymphocytes in those mice (see, for example, McCune et al. Science 241:1632-1639, 1988; Namikawa et al. J. Exp. Med. 172:1055-1063, 1990; Berenson et al. Blood 77:1717-1722, 1991).
Efforts have also been directed at developing an in vitro T-lymphopoiesis system (see, for example, Benveniste et al. Cell. Immunol. 127:92-104; Peault et al. J. Exp. Med. 174:1283-1286, 1991; Toki et al. Proc. natl. Acad. Sci. USA 88:7548-7551, 1991; Tjonnfjord et al. J. Exp. Med. 177:1531-1539; Hurwitz, J. Immunol. 17:751-756, 1987). Peault et al. have developed a system in which CD34+ cells from human fetal liver and bone marrow are microinjected in vitro into HLA-mismatched fetal thymus fragments previously depleted of hematopoietic stem cells by low temperature culture. Peault et al. have not achieved complete in vitro T-cell development, however, as the in vitro-colonized thymuses were subsequently engrafted into SCID mice, and T-cell differentiation occurred in vivo.
Tjonnfjord et al. have reported in vitro T-cell differentiation from adult human CD34+ bone marrow cells cultured in thymic stromal cell supernatant and in the presence or absence of recombinant murine c-kit ligand. Nevertheless, Tjonnfjord et al. found that only a small fraction of their total cell culture (see FIG. 3 of Tjonnfjord et al.) developed into mature T-cells, as indicated by the presence of T-cell-specific surface markers such as CD2, CD3, CD4, and CD8. Thus, there remains a need for the development of an in vitro T-lymphopoiesis system in which a significant fraction of the cultured cells develop into mature T-cells.